Optimal induction of p53 protein following DNA damage requires RPL26-mediated increases

Optimal induction of p53 protein following DNA damage requires RPL26-mediated increases in p53 mRNA translation. p53-mediated cell death after several different types of DNA damage and cellular stress. The ability to reduce stress induction of p53 with oligonucleotides or additional small molecules offers numerous potential restorative uses. mutations prospects to a serious susceptibility to malignancy development (Malkin et al. 1990; Levine 1997; Olivier et al. 2010). p53 protein transcriptionally regulates a complex network of genes involved in tumor suppression rate of metabolism autophagy and various physiological and pathological processes (Levine and Oren 2009). Degrees of p53 proteins upsurge in cells pursuing contact with DNA harm and other strains leading to mobile responses such as for VX-689 VX-689 example cell routine arrest or designed cell loss of life (Kastan and Bartek 2004). An capability to modulate tension induction of p53 in VX-689 tumor cells or regular cells could end up being useful in a variety of clinical settings. Boosts in p53 proteins amounts after DNA harm have been generally attributed to boosts in the half-life of p53 proteins. Proteasome-mediated degradation of p53 proteins regulated with the E3-ubiquitin ligase Mdm2 assists maintain low degrees of p53 proteins in nonstressed cells (Haupt et al. 1997; Honda et al. 1997; Kubbutat et al. 1997). Cellular exposure VX-689 to DNA damage results in an inhibition of Mdm2-mediated degradation of p53 protein contributing to an increase in p53 protein half-life and improved cellular levels of p53 protein (Ashcroft and Vousden 1999; Wade et al. 2010). We reported recently that improved translation of p53 mRNA is also a requisite step for ideal p53 induction following DNA damage (Takagi et al. 2005). In particular we found that RPL26 protein binds to the 5′-untranslated region (UTR) of p53 mRNA after DNA damage enhances the association of p53 mRNA with weighty polysomes and increases the translation of p53 mRNA. Furthermore the Mdm2-p53 opinions loop which regulates p53 levels after DNA damage (Levine 1997; Prives 1998; Michael and Oren 2003) also appears to involve Mdm2-RPL26 relationships which impact p53 translation (Ofir-Rosenfeld et al. 2008). Down-regulation of RPL26 protein levels with VX-689 siRNA not only reduces the raises in p53 translation seen after DNA damage it also reduces raises in total p53 protein levels and cell death after DNA damage (Takagi et al. 2005). Therefore blockade of RPL26-mediated translational induction of p53 is sufficient to reduce p53 induction and p53-mediated cell death. We report here that translation of human being p53 mRNA is definitely regulated by base-pairing relationships between 5′- and 3′-UTR sequences. RHOH12 Mutations that disrupt the connection abolish the binding of RPL26 to human being p53 mRNA and diminish RPL26-dependent p53 induction. Compensatory mutations that restore this UTR connection save the RPL26 binding as well as translational rules of p53 by RPL26. Small single-strand oligonucleotides comprising critical sequences from this RNA connection region inhibit the binding of RPL26 to p53 mRNA and blunt p53 induction after exposure of cells to a variety of DNA-damaging providers. These observations demonstrate a novel mechanism of regulating cellular protein levels and provide a reagent (oligonucleotides) that can be used to modulate stress induction of p53 in cells-a potentially clinically useful treatment. These mechanistic insights also arranged the stage for identifying small molecules other than oligonucleotides that could modulate p53 induction by focusing on this mechanism. Results p53 mRNA consists of a dsRNA structure with base-pairing between the 5′- and 3′-UTRs RPL26 activation of p53 translation requires the 5′-UTR but not the coding sequence of p53 mRNA (Takagi et al. 2005; Ofir-Rosenfeld et al. 2008). This 5′-UTR translational dependence was recapitulated by a cell-based dual-luciferase reporter assay in which RPL26 selectively enhanced the expression level of a chimera firefly luciferase reporter gene comprising a 5′-UTR sequence of human being p53 mRNA relative to the manifestation of the internal control renilla luciferase gene (Fig. 1A)..